The invention is directed to the determination of relative binding affinities of various ligands to various nucleic acid sequences, and in particular to the determination of binding specificities and base pair determinants of specificity of particular ligands via a competitive binding assay.
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The specific molecular recognition of nucleic acids is fundamental to essential processes in molecular biology, including replication, transcription and translation. It has been shown that, in the majority of cases, binding of ligands to double-stranded nucleic acids stabilizes the duplex, or helical, form of DNA or RNA. (See, for example, Wilson et al.) The current understanding of the interactions between DNA or RNA and bound ligands is largely based on information obtained via biochemical and biophysical methods such as chemical and nuclease footprinting, affinity probing, UV, CD, fluorescent, and NMR spectroscopy, calorimetry, gel electrophoresis, and x-ray crystallography.
In a typical application of DNA footprinting, for example, a labeled oligonucleotide is digested with a DNA nuclease to the extent necessary to create an average of one cut per chain, producing a series of fragments differing by one base pair in length. A similar operation is performed on the oligonucleotide having a bound ligand. The ligand protects the oligonucleotide, at and around its binding site, from nuclease activity, creating a characteristic pattern of xe2x80x9cmissingxe2x80x9d fragments at this site on a polyacrylamide gel following electrophoresis. This method suffers from the disadvantages of being very time and labor intensive, and in revealing not necessarily the critical molecular determinants for the ligand binding, but rather the area of the oligonucleotide that is shielded by the bulk of the ligand.
The most widely used method for studying nucleic acid hybridization is thermal denaturation, or melting, of duplex nucleic acids. Ligand binding has also been studied using thermal denaturation, since binding of ligands to duplex DNA or RNA tends to stabilize the helix against melting. Techniques used to observe this change include UV, fluorescent, CD and NMR spectroscopy, electrophoresis, and calorimetry.
Certain disadvantages are inherent in ligand binding studies based on observation of duplex denaturation, or melting. The methods provide information about binding only at or near the Tm of the system, rather than at standard (25xc2x0 C.) or physiological (37xc2x0 C.) temperatures. Because the presence of the ligand generally raises the Tm of the duplex, it is necessary that the ligand, e.g. the drug, be stable at this higher temperature. In addition, these methods do not routinely provide information about the binding site of the ligand (see, for example, Chen et al., 1997; Wilson et al., 1997). Therefore, the need exists for assays which are sensitive, are rapidly and simply carried out, and provide precise binding site information.
In one aspect, the invention provides a method of determining the binding affinity of a ligand to an oligonucleotide sequence. The ligand may be any of a wide range of nucleic acid binding groups, including a metal ion, a small organic molecule, a protein, a multi-protein complex, or a polynucleotide. According to the method, a mixture is formed of first and second oligonucleotides. The first oligonucleotide comprises a first group which is effective to produce a detectable signal, and the second oligonucleotide, which is effective to hybridize with the first oligonucleotide by Watson-Crick base pairing, comprises a second group which is effective to detectably alter the signal when the first and second oligonucleotides hybridize to form a duplex. In accordance with a preferred embodiment, the mixture is formed under conditions such that, in the absence of the ligand, the two oligonucleotides exist primarily in single-stranded form. The detectable signal, or lack thereof, is then observed in the absence of the ligand and in the presence of the ligand. Preferably, the ligand is added in increasing concentrations, with the mixture held at a substantially constant temperature, which may be at or near room temperature, during the addition. By titrating in the ligand and observing the change in signal, apparent Kd can be determined for any oligonucleotide sequence.
In various embodiments of the first and second groups, the second group is effective to stimulate or magnify the signal, to reduce or quench the signal, or to otherwise modify the signal upon hybridization of the first and second oligonucleotides. For example, where the signal is emitted radiation, e.g. from a fluorescent dye, the second group may be effective to quench the radiation, or to alter the wavelength of the radiation, by absorption and reemission, upon hybridization. In another embodiment, the first group is a radioactive emitting group, and the second group comprises a scintillant. Alternatively, the first group or the second group is a chemiluminescent group.
In one embodiment, the first group is attached at the 5xe2x80x2-end or 3xe2x80x2-end of the first oligonucleotide, and the second group is attached at the 3xe2x80x2-end or 5xe2x80x2-end, respectively, of the second oligonucleotide. Alternatively, the first and second groups can be attached at any position within the oligonucleotide.
In another aspect, the invention provides a related method of determining the binding affinity of a ligand to an oligonucleotide sequence. In accordance with this method, a duplex is formed of a first oligonucleotide, comprising a first group effective to produce a detectable signal, and a second oligonucleotide, effective to hybridize with the first oligonucleotide by Watson-Crick base pairing, and comprising a second group effective to detectably alter the signal when the first and second oligonucleotides hybridize. In this method, the first and second oligonucleotides differ in length, such that the resulting duplex has an overhang region, preferably about 4-7 nucleotides in length. An unlabeled displacement strand, which is effective to displace one of the oligonucleotides from the duplex in the absence of the ligand, is then added. The effect of this addition on the signal is observed in the absence and in the presence of the ligand. In a preferred embodiment, the forming, adding and observing steps are carried out at a substantially constant temperature, which may be at or near room temperature.
This method may further include the steps of adding a competitor oligonucleotide and observing the effect of such addition on the signal. The competitor oligonucleotide may be a duplex DNA, duplex RNA, duplex DNA/RNA hybrid, or a single stranded oligonucleotide.
In a further embodiment, the invention provides a method of determining the relative binding affinities of a ligand to different oligonucleotide sequences. The ligand may be, for example, a metal ion, a small organic or inorganic molecule, a protein, a multi-protein complex, or a polynucleotide. Accordingly, there is provided a mixture of a first oligonucleotide, which comprises a first group effective to emit a detectable signal, and a second oligonucleotide, effective to hybridize with the first sequence by Watson-Crick base pairing, which comprises a second group effective to detectably alter the signal when the first and second oligonucleotides hybridize. An indicator duplex of the two oligonucleotides, having the ligand bound thereto, is formed. A competitor oligonucleotide is then added, and the effect of this addition on the signal is observed. The competitor oligonucleotide is generally unlabeled, and may be a duplex DNA, duplex RNA, duplex DNA/RNA hybrid, or a single stranded oligonucleotide.
In a preferred embodiment, the indicator duplex is formed under conditions such that, in the absence of the ligand, the first and second oligonucleotides would exist primarily in single-stranded form. Preferably, the adding and observing steps are carried out at a substantially constant temperature, which may be at or near room temperature.
As above, the first group is preferably attached at the 5xe2x80x2-end or 3xe2x80x2-end of the first oligonucleotide, and the second group at the 3xe2x80x2-end or 5xe2x80x2-end, respectively, of the second oligonucleotide. In various embodiments, the first and second groups are, respectively, a radioactive emitting group and a scintillant, groups effective to produce a chemiluminescent reaction, or a fluorescent group and a group effective to absorb radiation emitted by the fluorescent group.